1. Field of the Invention
This invention relates to a novel bipartite plant viral vector launch system for recombination in planta to regenerate full-length infectious virus, a viral vector advantageous for high level replication for protein expression resulting from efficient suppression of RNA silencing and also advantageous for effective virus-induced gene silencing (VIGS) when modified to incorporate weak suppression of RNA silencing, cloned from a potexvirus, Alternanthera mosaic virus (AltMV); constructs containing the required parts of the bipartite viral vectors, constructs containing the RNA-dependent RNA polymerase of AltMV infectious clones, constructs comprising the partial RdRp, TGB1, TGB2, TGB3 and CP of AltMV infectious clones, and such constructs further comprising T7 promoters, Cauliflower mosaic virus (CaMV) 35S promoters, duplicated sub-genomic RNA promoters and multiple cloning sites allowing for additional genes/sequences for expression; vectors containing exogenous T7 RNA polymerase for enhancing infectivity, variant constructs containing modifications to the efficiency of RNA silencing suppression thus enabling VIGS, a method of making a novel bipartite plant viral vector launch system comprising constructs capable of regenerating full-length infectious virus vectors advantageous for both protein expression and VIGS of exogenous genes and sequences, a method of using both the CaMV 35S promoter and the T7 promoters in the same vector making possible both DNA and RNA delivery to drive the transient expression of the infectious viral clones for efficient protein expression and VIGS, a method of ensuring early and enhanced levels of infectivity by including the addition of vectors encoding T7 RNA polymerase, and a method of manipulating the severity of infection by altering the temperature.
2. Description of the Relevant Art
Alternanthera mosaic virus (AltMV) is a potexvirus most closely related to Papaya mosaic virus, and was originally reported from a weed species, Alternanthera pungens, in Australia (Geering and Thomas. 1999. Arch. Virol. 144:577-592). AltMV has since been reported to infect various ornamental plants, including several Phlox species (Hammond et al. 2006a. Arch. Virol. 151:477-493; Hammond et al. 2006b. Acta Horticulturae 722:71-77), and Portulaca (Ciuffo and Turina. 2004. Plant Pathol. 53:515; Hammond et al. 2006a,b, supra; Baker et al. 2006. Plant Disease 90:833), Scutellaria and Crossandra (Baker et al., supra), Angelonia (Lockhart and Daughtrey. 2008. Plant Dis. 92:1473) as well as Arabidopsis and soybean (H-S. Lim and J. Hammond, unpublished). The full 6607 nt sequence of one phlox isolate, AltMV-PA, has been reported (GenBank accession no. AY863024), and has a 5′ untranslated region (UTR) of 94 nt and a 126 nt 3′ UTR excluding the poly(A) tract; there are five open reading frames which encode the RNA-dependent RNA polymerase (RdRp), the triple gene block (TGB) proteins (TGB1, TGB2, TGB3), and the coat protein (CP) (Hammond at al. 2006b, supra). Some differences in symptom expression between isolates have been reported; in Nicotiana benthamiana, AltMV-SP (from phlox) and AltMV-PA produce faint chlorotic lesions on the inoculated leaves, followed by systemic chlorotic mosaic, with some rugosity and distortion, while AltMV-Po (from portulaca) produced mild symptoms. Under low temperature conditions (<20° C.) AltMV-SP produces necrotic local lesions and severe systemic necrosis (Hammond et al. 2006a, supra) an apparent hypersensitive response. It has also been noted that two geographically distinct isolates from portulaca share several coat protein (CP) amino acid residues that distinguish them from phlox and alternanthera isolates (Hammond et al. 2006a, supra); the complete sequence of a third geographically distinct isolate from portulaca has recently become available in GenBank (FJ822136) and the CP amino acid sequence is almost identical to those of the other portulaca isolates.
The presence of multiple sequence types, or mixed infection of distinct isolates, within a single plant has been reported for several viruses of different taxonomic groups e.g. capilloviruses (Magome et al. 1997; Phytopathology 87:389-396); closteroviruses (Sentandreu et al. 2006. Arch. Virol. 151:875-894); potyviruses (Sáenz et al. 2001. Mol. Plant Microbe Interact. 14: 278-287); alfalfa mosaic virus (Hull and Plaskitt. 1970. Virology 42: 773-776), including potexviruses (e.g. Ozeki et al. 2006. Arch. Virol. 151: 2067-2075). As RNA-dependent RNA polymerase lacks a proof-reading activity, RNA virus populations tend to accumulate many minor variations around a master sequence (or multiple master sequences), and thus exist as quasispecies populations (Domingo et al. 1985. Gene 40:1-8). Variation within isolates arises by viral polymerase error at a constant rate, but many of the newly generated mutants are sequestered in virions and may not serve as replication templates (Hall et al. 2001. J. Virol. 75: 10231-10243). However, the extent of population variation is limited by selection pressure for variants that interact successfully with different host and viral proteins necessary for completion of the infection cycle (Garcia-Arenal et al. 2001. Annu. Rev. Phytopathol. 39:157-186; Schneider and Roossinck. 2001. J. Virol. 75: 6566-6571; Rico et al. 2006. J. Virol. 80: 8124-8132). In order to survive, a virus must be diverse enough to adapt rapidly to changing environments without losing fitness during passage from host to host (Liang et al. 2002. J. Virol. 76: 12320-12324). Changing environmental conditions such as temperature can affect virus RNA replication (e.g. Aldaoud et al. 1989. Intervirology 30:227-233; Kaper et al. 1995. Arch. Virol. 140: 65-74). Genetic exchange plays a role to produce population diversity in bipartite or tripartite viruses (Lin et al. 2004. J. Virol. 78:6666-6675); but recombination has also been reported within monopartite viruses such as the potyviruses (Revers et al. 1996. J. Gen. Virol. 77: 1953-1965), closteroviruses (Rubio et al. 2001. J. Virol. 75: 8054-8062), and between defective potyviral genomes delivered as RNA transcripts (Gal-On et al. 1998; J. Virol. 72:5268-5270). There is also some evidence for recombination between isolates of the potexvirus Cymbidium mosaic virus (Sherpa et al. 2007. J. Biosci. 32: 663-669; Vaughan et al. 2008. Arch. Virol. 153: 1186-1189), although others found no such evidence (Moles et al. 2007. Arch. Virol. 152: 705-715), and Malcuit et al. (2000, Virus Genes 20: 165-172) have suggested that PVX strain groups evolved through convergent evolution rather than recombination. More recently Draghici and Varrelmann (2009. J. Virol 83: 7761-7769) have demonstrated RNA recombination under high selective pressure between defective PVX genomes delivered by agroinfiltration; in this instance the defective genomes either lacked the 5′ or the 3′ viral untranslated regions (utr) such that no replication of either partial genome alone was possible. Selection may disfavor recombined strains, as a result of incompatibilities between interacting viral proteins, or between viral proteins and cis-acting viral sequences (Malcuit et al., supra). The derivation of distinct Citrus tristeza virus (CTV) lineages by evolution and/or selection from a quasispecies population aided by host passage or aphid transmission has been documented (Sentandreu et al., supra). The origin of variants from a population derived from infectious cDNA clones of Tobacco mosaic virus (Kearney et al. 1999. Arch. Virol. 144: 1513-1526; Schneider and Roossinck. 2000. J. Virol. 74:3130-3134), Cucumber mosaic virus (CMV) and Cowpea chlorotic mottle virus (Schneider and Roossinck, 2000, supra) has been documented, as has selection from a defined population of CMV variants (Li and Roossinck. 2004. J. Virol. 78:10582-10587).
The Potexvirus replicase is a single protein that contains methyltransferase, RNA helicase and RNA polymerase domains (Verchot-Lubicz et al. 2007. J. Gen. Virol. 88:1643-1655). A single amino acid change in the Pol domain of Potato virus X (PVX) or Plantago asiatica mosaic virus (PIAMV) RdRp induces systemic necrosis in N. benthamiana (Kagiwada et al. 2005. Virus. Res. 110:177-182; Ozeki et al., supra). PVX TGB1 has been reported as a suppressor of RNA silencing (Bayne et al. 2005. Plant J. 44: 471-482; Voinnet et al. 2000. Cell 103:157-167), and to block systemic spread of the silencing signal (Voinnet at al., supra). TGB1, TGB2, TGB3 and coat protein are required for movement; TGB2 and TGB3 are ER binding proteins, and TGB2 has two transmembrane domains and a central motif conserved'among potexviruses that lies in the ER lumen (Verchot-Lubicz at al., supra). PVX CP is an elicitor of the Rx resistance response, and overproduction of CP leads to Rx-mediated hypersensitive response (Bendahmane et al. 1995. Plant J. 8: 933-941; Tameling and Baulcombe. 2007. Plant Cell 19:1682-1694).
A strategy for use of RNA plant viruses as vectors was proposed by Siegel (Siegel, A. 1983. Phytopathology 73: 775) even before infectious clones of any plant viruses had been developed. According to this strategy, the rod-shaped plant viruses offered the greatest possibility as vectors, because the architecture of the particles does not place inherent limitations on the size of the insert; in contrast there are clear packaging constraints with viruses that have isometric particles. Several types of viral vectors have been developed among the rod-shaped and filamentous plant viruses, including gene replacement vectors, exemplified with Tobacco mosaic virus (TMV) by Takamatsu et al. (1987. EMBO J. 6: 307-311); gene insertion behind a duplicated subgenomic promoter from the same virus (Dawson et al. 1989. Virology 172: 285-292) or a related virus (Culver et al. 1993. Proc. Natl. Acad. Sci. USA 90: 2055-2059); translational fusions of partial or complete ORFs to either the N-terminus or C-terminus of a viral structural protein, either with or without a proteolytic cleavage site allowing processing of the fused sequence (e.g. Gopinath et al. 2000. Virology 267: 159-173), as a readthrough fusion such that both wild-type and modified viral proteins are produced (Hamamoto at al. 1993. Bio/Technology 11: 930-932); epitope display in an internal, surface-exposed loop (Porta at al. 1994. Virology 202: 949-955), functional complementation with multicomponent viruses such as Cucumber mosaic virus (Zhao et al. 2000. Arch. Virol. 145:2285-2295); functional complementation of a defective RNA by a wild-type virus (Raffo & Dawson. 1991. Virology 184: 277-289); chimeric viruses expressing a heterologous viral CP for peptide presentation or epitope display (Yusibov et al. 1997. Proc. Natl. Acad. Sci. USA 94: 5784-5788); and viral amplicons delivered from the genome of a transgenic plant (Angell & Baulcombe. 1997. EMBO J. 16: 3675-3684) or via agroinfiltration (Liu & Lomonossoff, 2002. J. Virol. Methods 105: 343-348). Mallory et al. (2002. Nat. Biotechnol. 20:622-625) reported the use of a viral suppressor of RNA silencing to overcome RNA silencing to increase expression from the viral amplicon. Knapp at al. (2005. Virology 341: 47-58) have developed a bipartite system from a defective genome of TMV lacking the CP gene, paired with a defective RNA having an internally deleted replicase gene and a functional CP gene flanked by the TMV 5′ and 3′ TMV utr. Although this bipartite form was maintained in systemic infections, systemic movement was significantly debilitated (Knapp et al. 2007. Virology 367:82-91).
Replication of defective genomes in the presence of a fully functional genome occurs in several plant viral systems; in some cases replication of a ‘defective interfering” (DI) RNA inhibits replication of the functional genome (e.g. Jones et al. 1990. Virology 176:539-545). In other cases the DI RNA may intensify symptom expression (e.g. Li et al. 1989. Proc Natl Acad Sci USA 86, 9173-9177).
A further extension of hybrid viruses, combined with complementation, has been described. Marillonet et al. (2004. Proc Natl Acad Sci USA 101: 6852-6857) and Gleba et al. (2004. Curr. Opin. Plant Biol. 7: 182-188) have developed systems in which viral functions not needed for expression can be dispensed with, and complementation used to provide equivalent functions from other sources; this has been described as the ‘deconstructed virus’ approach; expression can be optimized by elimination of functions not needed for expression. This may also aid in biocontainment, by elimination of functions contributing to vectored transmission. An efficient means of delivery of multiple separate components by agroinfiltration, combined with in planta recombination through co-expression of a recombinase, was shown to both confer high yields, and to allow greater flexibility in comparing variants of one or more system components. One or more component may be supplied as a transgene, and induction of replication and expression may be regulated by use of either a developmentally-controlled promoter, or by application of an inducer (Gleba et al. 2004, supra).
Taschner et al. (1991. Virology 181: 445-450) transformed plants with the replicase functions of Alfalfa mosaic virus (AlMV), while Mori et al (1992. J. Gen. Virol. 73: 169-172) similarly transformed plants with the replicase functions of Brome mosaic virus; Sanchez-Navarro et al. (2001. Arch. Virol. 146: 923-929) engineered RNA3 of AlMV into an expression vector using the replicase-expressing plants.
Virus-Induced Gene Silencing (VIGS) has become a significant tool for discovery of gene function in both dicotyledonous (Ratcliff of al. 2001. Plant J. 25: 237-245; Liu et al. 2002. Plant J. 31: 777-786) and monocotyledonous (Holzberg et al. 2002. Plant J. 30: 315-327) species. Although some viral vectors have been utilized for both protein expression and VIGS, including PVX (Chapman et al. 1992. Plant J. 2: 549-557; Ruiz of al. 1998, Plant Cell 10: 937-946) and Bean pod mottle virus (BPMV; Zhang & Ghabrial. 2006. Virology 344: 401-411), it is generally recognized that for high level protein expression, a virus with an effective suppressor of RNA silencing is desirable, whereas for VIGS, a less effective viral suppressor of RNA silencing is preferred (Dalmay of al. 2000. Plant Cell 12: 369-379). Indeed, it has been noted that BPMV does not effectively suppress RNA silencing, and that expression of an effective suppressor of RNA silencing in combination with BPMV vectors may be useful for enhancing foreign protein expression, which would probably need to be expressed from a co-infecting recombinant BPMV vector (Zhang and Ghabrial 2006, supra). BPMV has either weak or no suppressor of RNA silencing (Zhang and Ghabrial 2006, supra), and is more useful as a VIGS vector (Zhang et al. 2009. Mol. Plant—Microbe Interact. 22:123-131). PVX may fall into a middle group, as replication of PVX is significantly enhanced in a mixed infection with a potyvirus, as a consequence of the efficient suppression of RNA silencing provided by the potyvirus HC-Pro (Pruss et al. 1997. Plant Cell 9: 859-868), although PVX has its own suppressor of RNA silencing in TGB1 (Voinnet et al. 2000. Cell 103: 157-167).
Infectious clones of potexviruses including PVX (Hemenway et al. 1990. Virology 175: 365-371) have been developed and used as vectors for gene expression in plants (Chapman et al. 1992, supra) and for VIGS (Ruiz et al. 1998, supra). Separate constructs of infectious PVX clones driven by the Cauliflower mosaic virus (CaMV) 35S promoter and the bacteriophage T7 promoter have been reported (Baulcombe et al. 1995. Plant J. 7: 1045-1053). An infectious monopartite clone of PVX has been placed into a binary Agrobacterium vector under the control of the CaMV 35S promoter, and further modified with unique restriction enzymes in a Multiple Cloning Site (MCS) to allow high throughput cloning and expression of suitably constructed cDNA libraries (Takken et al. 2000. Plant J. 24: 275-283).
The bacteriophage T7 RNA polymerase (T7RNAP) is well known, and has previously been utilized from a chromosomal insertion for high-level expression of genes in bacteria (Studier & Moffatt. 1986. J Mol Biol. 189:113-130); in mammalian cells when T7RNAP was itself expressed from a recombinant vaccinia virus (Fuerst at al. 1986. Proc Natl Acad Sci USA. 83:8122-8126); and in insect cells when T7RNAP was delivered from a recombinant baculovirus (van Poelwijk et al. 1995. Biotechnology (NY). 13:261-264). The T7RNAP has also been used under the control of appropriate promoters for both tissue-specific and inducible expression in transgenic plants (Nguyen et al. 2004. Plant Biotechnol J 2, 301-310). Replication of the RNA of an insect virus was initiated (Ball. 1995. J. Virol. 69:720-727), and infectious rabies virus recovered in mammalian cells (Schnell et al. 1994. EMBO J. 13:4195-4203) using the vaccinia/T7 system. Infectious poliovirus was recovered from mammalian cells using the baculovirus/T7 system (Yap et al. 1997. Virology 231: 192-200). Reverse-genetics systems for negative-strand viruses are also often based on transcription of viral RNA by the bacteriophage T7 RNA polymerase (de Wit at al. 2007. J Gen Virol 88: 1281-1287). A hybrid baculovirus-T7 RNA polymerase system has been used for transient expression in mammalian cells (Yap et al. 1997, supra). The RNA minigenome system has been used for evaluating the functions of viral proteins and of sequences involved in viral RNA replication (Lohmann et al. 1999. Science 285: 110-113). Much of the work on the role of the 5′ and 3 UTR in viral replication has been through use of the minigenome (Dumas et al. 2007. J Virol Methods 142: 59-66; Friebe at al. 2001. J. Virol. 75: 12047-12057).
In summary, agroinfiltration, biolistic delivery of plasmid or transcripts, and mechanical inoculation of either plasmids or in vitro transcripts are the main methods for delivery of viral nucleic acids into cells. Agroinfiltration is relatively easy to apply with low cost; however, not all plant species are susceptible to agroinfiltration. In contrast, most plant species can be infected by biolistic delivery or mechanical inoculation with plasmids or in vitro transcripts, but the technique is more laborious and expensive than agroinfiltration. Each method has advantages, and disadvantages, but since most virus-based vectors are constructed with a single promoter sequence, it has not typically been possible to use both DNA and RNA delivery methods with the same vector. In addition, many plant viruses have been modified, for either protein expression or gene silencing. However, a single plant virus-based vector has been unable to effectively fulfill both functions because of the conflicting requirements for strong or weak RNA silencing suppression, respectively, for protein expression and VIGS.
Thus, there is a need for the development of alternative viral vector systems which have flexibility for modification and variation and which are applicable for effective in planta transient expression of multiple genes. We show here that the genome of representative potexviruses can be manipulated to serve as new bipartite plant viral vector launch systems for recombination in planta to regenerate full length infectious viruses, viral vectors effective for high levels of protein expression, and manipulated differently, for effective virus-induced gene silencing.